Title: Purple Protein Expression, Purification, and Characterization
Introduction:
There are many useful applications for using recombinant proteins that have been steadily increasing over the years. As more techniques are fine-tuned and developed, the scientific community has been better able to purify, amplify and study desired proteins. E. coli is a commonly used bacterium due to our extensive knowledge of it's systems and it's easy ability to be used in protein expression [1]. With the use of enzymes, genes can be cloned and expressed in various vectors which can then be amplified in bacterial cells. After extraction of the proteins, techniques such as chromatography can be used to purify the sample and gel electrophoresis can be used to study the sample [2].
In this series of three labs, the goal was to express, purify and characterize the fluorescent protein gbr22 using E. coli vectors. If the sample in the experiment is purified and characterized correctly, the protein should be visualized as a dark band of specific weight on the gel electrophoresis and weigh in around 25 kDa.
Materials & Methods: This three part, multi-day lab, required various materials including: ice bucket, gas burner, two 14 ml clear, sterile round-bottom transformation tubes, 37°C incubator, colirollers, two LB Agar Amp plates, one spare Agar plate without antibiotics, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipettes, pipette tips, 1M imidazole, 10x PBS, 1x PBS, 1.7 ml centrifuge tubes, one 14 ml round bottom transformation tube, two 10 ml round bottom tubes, four 15 ml conical tubes, Bio-Rad Econo chromatography column with yellow cap and clear round top, ring stand and clamps, Ni-NTA resin, cyanase (benzonase), heat block, mini-PROTEAN electrophoresis tank and lid, TGS running buffer, Bio-Rad precast polyacrylamide gel, 6x gel loading buffer, plastic container and imperial protein stain.
This three part lab took place over the course of multiple days and required the results of the previous steps. First, three plates need to be created, one experimental one with ampicillin, pGEM-gbr22 plasmid vector and BL21(DE3 ) bacteria (New England BioLabs, Ipswich, MA); one with ampicillin, BL21(DE3) bacteria and no DNA plasmid; and finally a fun plate. Using bacterial transformation techniques, the plates should be placed for 30 minutes on ice then heat shocked in 42°C water for 45 seconds and placed on ice for an additional two minutes. After adding 200 ul of SOC media shake in an incubator at 37°C for 30 minutes. Then spread bacteria over plates and store in 37°C incubator overnight. The next morning, pick a single colony of bacteria to drop in a tube of LB and place tube in incubator for 8 hours at 37°C. A large culture was then created by transferring the starter culture to a flask that was then place in a 37°C incubator for 16-24 hours. After words, cells were harvested and suspended in a PBS solution. Lysozyme was then added and the tube was placed in the -20°C freezer.
Purifying the sample required thawing the tubes placed in the freezer then adding cyanase and centrifuging for 20 minutes at 14,000 rpm and 4°C. After centrifugation, sample two was collected and the wash and elution buffers were created. Ni-NTA was then added to the chromatography column and the lysate was allowed to flow through and sample three was taken. The column was washed with 1x PBS to obtain sample 4, then washed again with the imidazole buffer to obtain elution 1 (sample 5). The lysate was washed again to obtain elution 2 (sample 6) and the samples were stored in the 4°C freezer. The column was then stripped down to allow for reuse. The Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was then used to measure the absorbance of elution 1 at two different wavelengths. Characterization was carried out through the loading and running of samples 1-6 on an SDS-PAGE gel electrophoresis. The gel was then stained, dried, and compared to the molecular weight standards.
Results:
Figure 1a: Ampicillin and LB control agar plate, plated with ampicillin, BL21(DE3) bacteria and no DNA plamid. There is no visible bacterial growth after 24 hours incubation at 37 degrees Celsius.
Figure 1: LB agar plate, plated with bacteria gathered from wiping down a cellphone--"fun plate". There is no visible bacterial growth after 24 hours incubation at 37 degrees Celsius.
Figure 1c: Ampicillin and LB agar plate, plated with ampicillin, BL21(DE3) bacteria and DNA plamid pGEM-gbr22. There is extensive bacterial growth visible after 24 hours incubation at 37 degrees Celsius.
Figure 2: A large culture of BL21(DE3) bacteria transformed with the plasmid DNA pGEM-gbr22, seen in an Erlenmeyer flask, after cultivating a single bacterial colony from Figure 1c in LB broth and ampicilin.
Figure 3: A wet pellet of 0.53g obtained from centrifuging the culture of BL21(DE3) bacteria transformed with plasmid DNA pGEM-gbr22 as seen in Figure 2.
Figure 4: 14ml conical tube containing contents of Elution 1.
Figure 5: 14ml conical tube containing contents of Elution 2.
Figure 6a: Absorbance vs. Wavelength of Elution 1 at 280nm wavelength, Trial 1.
Figure 7. Dried SDS-PAGE gel with samples 3-8 loaded in lanethe molecular weight standard can be seen in lane 2, and a different set of samples 4-6 in lanes 9-10. This dried gel is seen in the picture upside down and reversed with lane 1 starting on the left and moving towards the right.
Figure 8: SDS-PAGE band profile of Thermo Scientific PageRuler Pre-Stained protein ladder depicting molecular weights in kDa.
Discussion: Lysozyme was added to that the bacterial cells would break open and allow the protein (which was expressed inside the cell) to be released and available for purification. Benzonase/Cyanase was used to degrade any bacterial DNA so that purer samples of the protein could be obtained.
The histidinne tags work to separate the gbr22 protein due to its affinity to bind to the Ni-NTA. Imidazole competes with the HIS residues and allowed other proteins to be washed away initially, and then after the second wash at a higher concentration, allowed for the gbr22 protein to be released.
Sample 1 contains a mixture of both the gbr22 protein and other proteins and molecules that made up the cell. Sample 2 was the soluble part of the solution and no longer contained DNA/RNA as well as the majority of the excess cellular debris that was not wanted. Sample 3 was taken after being flowed through the NI-NTA chromatography and held the proteins that failed to bind during the wash. Sample 4 had the contents of the solution after being washed while sample 5 contained the contents after the elution buffer was run through the column a second time. This sample shoud have contained the gbr22 protein and appeared as a dark band in the gel lane. Sample 6 contained gbr22 protein after the elution buffer was once again run through the chromatography column and also appeared as a slightly lighter band.
The wash and elution samples are different due to their contents. The wash sample contains all the loose proteins that failed to successful bind to the Ni-NTA in the chromatography column. These proteins could potentially be the gbr22, but they are likely other proteins that had yet to be separated from the solution. The elution on the other hand contains all the loosened gbr22 proteins that were released by the imidazole from their bonds.
The gel that ran depicted three strong dark bands which lends to the idea of possible contamination. While all three bands were at varying weights, one strong band appeared around the 24 to 25 kDa mark which is about the size of the gbr22 protein. While some contamination did occur, the band seen is likely the desired protein.
The sample as seen from the results was not a pure as would have been preferred. Judging by the size of the two other bands, the sample was about 33% pure. A large source of error was likely contamination that could have occurred along numerous steps of this process.
Conclusions:
While the gbr22 protein was expressed successfully in the E. coli bacterial cells, the purification was not as ideal as it should have been. The gbr22 protein was first transformed in bacterial cells to obtain amplified expression. After expression, the protein was purified through the use of an Ni-NTA chromatography column and characterization was done through the use of an SDS-PAGE. In the future, these techniques can be used to study other proteins or targets of interests which may help in various fields of medical research. In relation to VDS research, these techniques will be applicable in the investigation of protein drug targets to be chosen later in the future.
References:
[1] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards,A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.;Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.;Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.;Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.;Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.
Introduction:
There are many useful applications for using recombinant proteins that have been steadily increasing over the years. As more techniques are fine-tuned and developed, the scientific community has been better able to purify, amplify and study desired proteins. E. coli is a commonly used bacterium due to our extensive knowledge of it's systems and it's easy ability to be used in protein expression [1]. With the use of enzymes, genes can be cloned and expressed in various vectors which can then be amplified in bacterial cells. After extraction of the proteins, techniques such as chromatography can be used to purify the sample and gel electrophoresis can be used to study the sample [2].
In this series of three labs, the goal was to express, purify and characterize the fluorescent protein gbr22 using E. coli vectors. If the sample in the experiment is purified and characterized correctly, the protein should be visualized as a dark band of specific weight on the gel electrophoresis and weigh in around 25 kDa.
Materials & Methods:
This three part, multi-day lab, required various materials including: ice bucket, gas burner, two 14 ml clear, sterile round-bottom transformation tubes, 37°C incubator, colirollers, two LB Agar Amp plates, one spare Agar plate without antibiotics, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipettes, pipette tips, 1M imidazole, 10x PBS, 1x PBS, 1.7 ml centrifuge tubes, one 14 ml round bottom transformation tube, two 10 ml round bottom tubes, four 15 ml conical tubes, Bio-Rad Econo chromatography column with yellow cap and clear round top, ring stand and clamps, Ni-NTA resin, cyanase (benzonase), heat block, mini-PROTEAN electrophoresis tank and lid, TGS running buffer, Bio-Rad precast polyacrylamide gel, 6x gel loading buffer, plastic container and imperial protein stain.
This three part lab took place over the course of multiple days and required the results of the previous steps. First, three plates need to be created, one experimental one with ampicillin, pGEM-gbr22 plasmid vector and BL21(DE3 ) bacteria (New England BioLabs, Ipswich, MA); one with ampicillin, BL21(DE3) bacteria and no DNA plasmid; and finally a fun plate. Using bacterial transformation techniques, the plates should be placed for 30 minutes on ice then heat shocked in 42°C water for 45 seconds and placed on ice for an additional two minutes. After adding 200 ul of SOC media shake in an incubator at 37°C for 30 minutes. Then spread bacteria over plates and store in 37°C incubator overnight. The next morning, pick a single colony of bacteria to drop in a tube of LB and place tube in incubator for 8 hours at 37°C. A large culture was then created by transferring the starter culture to a flask that was then place in a 37°C incubator for 16-24 hours. After words, cells were harvested and suspended in a PBS solution. Lysozyme was then added and the tube was placed in the -20°C freezer.
Purifying the sample required thawing the tubes placed in the freezer then adding cyanase and centrifuging for 20 minutes at 14,000 rpm and 4°C. After centrifugation, sample two was collected and the wash and elution buffers were created. Ni-NTA was then added to the chromatography column and the lysate was allowed to flow through and sample three was taken. The column was washed with 1x PBS to obtain sample 4, then washed again with the imidazole buffer to obtain elution 1 (sample 5). The lysate was washed again to obtain elution 2 (sample 6) and the samples were stored in the 4°C freezer. The column was then stripped down to allow for reuse. The Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was then used to measure the absorbance of elution 1 at two different wavelengths.
Characterization was carried out through the loading and running of samples 1-6 on an SDS-PAGE gel electrophoresis. The gel was then stained, dried, and compared to the molecular weight standards.
Results:
Calculations:
Extinction coefficient (280 nm): 38850
Molecular Weight (gbr22): 25794.2
Beer's Law: A = εbc
0.158 = (38850)(c)
c = 0.0000041 mol/L
[(25794.2 g)/(1 mol)][(0.0000041 mol)/(1 L)][(1 L)/(1000 mL)][(100 mg)/(1 g)] =0.1049 mg/mL
Discussion:
Lysozyme was added to that the bacterial cells would break open and allow the protein (which was expressed inside the cell) to be released and available for purification. Benzonase/Cyanase was used to degrade any bacterial DNA so that purer samples of the protein could be obtained.
The histidinne tags work to separate the gbr22 protein due to its affinity to bind to the Ni-NTA. Imidazole competes with the HIS residues and allowed other proteins to be washed away initially, and then after the second wash at a higher concentration, allowed for the gbr22 protein to be released.
Sample 1 contains a mixture of both the gbr22 protein and other proteins and molecules that made up the cell. Sample 2 was the soluble part of the solution and no longer contained DNA/RNA as well as the majority of the excess cellular debris that was not wanted. Sample 3 was taken after being flowed through the NI-NTA chromatography and held the proteins that failed to bind during the wash. Sample 4 had the contents of the solution after being washed while sample 5 contained the contents after the elution buffer was run through the column a second time. This sample shoud have contained the gbr22 protein and appeared as a dark band in the gel lane. Sample 6 contained gbr22 protein after the elution buffer was once again run through the chromatography column and also appeared as a slightly lighter band.
The wash and elution samples are different due to their contents. The wash sample contains all the loose proteins that failed to successful bind to the Ni-NTA in the chromatography column. These proteins could potentially be the gbr22, but they are likely other proteins that had yet to be separated from the solution. The elution on the other hand contains all the loosened gbr22 proteins that were released by the imidazole from their bonds.
The gel that ran depicted three strong dark bands which lends to the idea of possible contamination. While all three bands were at varying weights, one strong band appeared around the 24 to 25 kDa mark which is about the size of the gbr22 protein. While some contamination did occur, the band seen is likely the desired protein.
The sample as seen from the results was not a pure as would have been preferred. Judging by the size of the two other bands, the sample was about 33% pure. A large source of error was likely contamination that could have occurred along numerous steps of this process.
Conclusions:
While the gbr22 protein was expressed successfully in the E. coli bacterial cells, the purification was not as ideal as it should have been. The gbr22 protein was first transformed in bacterial cells to obtain amplified expression. After expression, the protein was purified through the use of an Ni-NTA chromatography column and characterization was done through the use of an SDS-PAGE. In the future, these techniques can be used to study other proteins or targets of interests which may help in various fields of medical research. In relation to VDS research, these techniques will be applicable in the investigation of protein drug targets to be chosen later in the future.
References:
[1] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards,A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.;Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.;Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.;Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.;Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.
[2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/protein_purification/purification/index. (accessed April 17, 2013).